The major objective of this proposal is the biochemical identification and characterization of the primary defect in a group of mutants in culture which are pleiotropic for the utilization of alternate carbon sources in place of glucose. In contrast to the parental Chinese hamster lung cell line (V79), these mutants are unable to grow on galactose, fructose, glucose-6-phosphate, glucose-1-phosphate, galactose-1-phosphate, and some do not grow on mannose. The pleiotropic mutants appear to be caused by a mutation at a single locus and have been grouped into nine complementation units. Preliminary studies suggest that the pleiotropic carbohydrate mutants are defective in oxidative energy production due to a mutation affecting a citric acid cycle enzyme, component of the electron transport chain, or oxidative phosphorylation. These possibilities are being investigated in a systematic manner using purified mitochondria from mutants, wild type, revertants, and complementing cell hybrids. Biochemical characterization of the properties of the modified protein in the mutants or revertants will distinguish between a mutation in a regulatory gene controlling transcription or translation and a mutation in a gene affecting structure and/or activity. Hybridization of enucleated chloramphenicol resistant cells with the pleiotropic mutants will show if the mutation is cytoplasmically inherited.